Tools are needed to establish the etiology of Alzheimer?s disease (AD), whose signatures appear before the disease presents. Early in AD is ubiquitylation of misfolded proteins; those not degraded by the proteasome can form neurofibrillary tangles such as ?-amyloid or tau protein aggregates. Ubiquitin (Ub)-mediated signaling plays a central role in protein degradation, and a first step in neurodegeneration is a change in ubiquitylation patterns, particularly, that of poly-Ub chains attached to proteins. Development of sensitive methods to detect these ubiquitylation signatures will be a major advance in AD drug discovery and diagnosis. Poly-Ub chains are formed utilizing all seven lysines, as well as the N-terminus of ubiquitin, suggesting that these chains encode much information. Each linkage, including mono-Ub, signals a characteristic outcome for the protein; Ub chain architecture is the subject of this project. The best characterized Ub types are Lys48 (K48) and Lys63 (K63) linkages; K48- and K11-linked chains target proteins for degradation by the proteasome while K63-linked chains regulate receptor endocytosis, DNA repair, oxidative stress and other pathways. Specific functions for other linkages are currently under study. Two impediments to understanding specific poly-ubiquitylated protein structures and functions are: 1) while most proteins at some point are ubiquitylated, at any given time the ubiquitylated fraction may be a small percentage of total cell protein; and 2) within the pool of ubiquitylated protein, any particular linkage may be rare. Further, very few tools are available for specific detection or purification of ubiquitylated proteins with specific poly-Ub linkages. In phase I, Ub Binding Domains (UBDs) selective for atypical poly-Ub (K6/K11/K27/K29/K33) were identified using a 150 UBD protein microarray chip developed by Dr Mark Bedford, M D Anderson Cancer Center. Also identified was the UBD with the highest selectivity for K48 linked poly-ubiquitylated proteins. In addition, KD values and specificity for novel UBDs were determined using SPR, fulfilling the aims. In Phase II LifeSensors will engineer the UBD domains to improve affinity and selectivity using UBD domains called TUBEs (Tandem Ubiquitin Binding Entities); these include nonselective as well as Linear [M1], K48- and K63-specific TUBEs. Dr. Bedford will continue his collaboration in Phase II. Also participating in Phase II will be Dr. David Fushman, University of Maryland, who has pioneered solution NMR methods to study poly-Ub chains. the Phase II project entails establishing the poly-Ub chain linkage selectivity of UBDs identified in Phase I using structure-based protein engineering and generating poly-Ub chain- selective TUBEs to improve affinity, enhance avidity of binding, and improve linkage selectivity. Finally, these novel atypical poly-Ub TUBEs will be validated for application in neuronal cells by simulating proteinopathies. .